Immunization with live, replication-competent Ad-HIV or Ad-SIV envelope recombinant vaccines primes strong antibody responses that develop following administration of booster immunizations with envelope protein. These antibodies display a variety of functional activities. The most desirable for an HIV/AIDS vaccine is neutralizing activity that is able to prevent infection following exposure to the virus. We have elicited such antibodies in the rhesus macaque model that conferred apparent sterilizing immunity following challenge with an HIV/SIV chimeric SHIV virus. Our vaccine regimen also elicits antibodies with other functional activities mediated by Fc-receptor bearing cells such as NK cells. HIV/SIV infection is initially manifested as small foci of infected cells. Within 2 to 6 days, virus spreads from these cell foci to draining lymph nodes, subsequently leading to systemic infection. These additional functional activities can help control the initial viral burden by limiting the spread of virus from these foci of infection. Such activities include antibody dependent cellular cytotoxicity (ADCC), antibody dependent cell-mediated viral inhibition (ADCVI), and antibody-dependent cellular phagocytosis. Since HIV is transmitted mainly at rectal/genital mucosal sites, a key goal of HIV vaccine development is to elicit mucosal immunity. The Ad-recombinant prime/protein boost strategy induces antibodies in mucosal secretions which can inhibit transcytosis of SIV across an epithelial cell barrier, suggesting another mechanism which may contribute to protection. These mechanisms are all currently under study. The overall goal of vaccination is to develop immune memory. We have developed methodology to investigate memory B cells, which secrete antibodies. The ability of vaccines to elicit long lasting memory B cells is a critical property if immunization is to provide long-lasting, and potentially life-long protection. Over the past year, we have focused extensively on B cell development and maturation, and vaccine-elicited antibody responses, both systemic and mucosal. We investigated memory B cells in mucosal tissues of macaques which had not been previously characterized. We phenotyped the cells, and developed a panel of specific markers to definitively identify long-lived antibody-secreting plasma cells from shorter lived plasma blasts. These findings will facilitate further investigations of vaccine-elicited antibody responses. We also developed an improved flow cytometry-based method to identify viral envelope specific memory B cells, again an advancement which will enhance our understanding of vaccine-induced humoral immunity and immune correlates of protection. We have also investigated marginal zone B cells in secondary lymphoid tissue of macaques, furthering our understanding of B cell development and maturation and dysfunction that occurs following SIV infection. We demonstrated that mucosal tissue (rectal) can be directly cultured short-term allowing collection of mucosal immunoglobulin for further studies and quantitation. A study in the rhesus macaque model compared a number of mucosal and systemic vaccine regimens, and showed that mucosal priming with our replication-competent Ad-recombinant strategy elicits early mucosal antibody responses, allowing us to conclude that a combined mucosal/systemic vaccine strategy may improve vaccine protective efficacy. This strategy will be explored in future pre-clinical vaccine studies. We have developed methods to purify large quantities of secretory IgA from vaccinated or infected macaques which will allow further characterization of potentially protective activities. We have also initiated studies of the effector cells that mediate antibody-dependent activities. On-going studies are examining factors in bone marrow and mucosal tissues which recruit and maintain plasma cells at the sites.